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Long-distance transport along microtubules (MTs) is critical for intracellular organization. In animals, antagonistic motor proteins kinesin (plus end directed) and dynein (minus end directed) drive cargo transport. In land plants, however, the identity of motors responsible for transport is poorly understood, as genes encoding cytoplasmic dynein are absent in plant genomes. How other functions of dynein are brought about in plants also remains unknown. Here, we show that a subclass of the kinesin-14 family, KCH (kinesin with calponin homology domain), which can also bind actin, drives MT minus enddirected nuclear transport in the moss Physcomitrella patens. When all four KCH genes were deleted, the nucleus was not maintained in the cell center but was translocated to the apical end of protonemal cells. In the knockout (KO) line, apical cell tip growth was also severely suppressed. KCH was localized to MTs, including at the MT focal point near the tip of protonemal cells, where MT plus ends coalesced with actin filaments. MT focus was not stably maintained in KCH KO lines, whereas actin destabilization also disrupted the MT focus in wild-type lines despite KCH remaining on unfocused MTs. KCH had distinct functions in nuclear transport and tip growth, as a truncated KCH construct restored nuclear transport activity, but not tip growth retardation of the KO line. Thus, our study identified KCH as a long-distance retrograde transporter as well as a MT cross-linker, reminiscent of the versatile animal dynein.

In textbooks, the mitotic spindles of plants are often described separately from those of animals. How do they differ at the molecular and mechanistic levels? In this chapter, we first outline the process of mitotic spindle assembly in animals and land plants. We next discuss the conservation of spindle assembly factors based on database searches. Searches of >100 animal spindle assembly factors showed that the genes involved in this process are well conserved in plants, with the exception of two major missing elements: centrosomal components and subunits/regulators of the cytoplasmic dynein complex. We then describe the spindle and phragmoplast assembly mechanisms based on the data obtained from robust gene loss-of-function analyses using RNA interference (RNAi) or mutant plants. Finally, we discuss future research prospects of plant spindles.

Kinesin-13 and Kinesin-8 are well-known microtubule (MT) depolymerases that regulate MT length and chromosome movement in animal mitosis. While much is unknown about plant Kinesin-8, Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) Kinesin-13 have been shown to depolymerize MTs in vitro. However, the mitotic function of both kinesins has yet to be determined in plants. Here, we generated complete null mutants of Kinesin-13 and Kinesin-8 in moss (Physcomitrella patens). Both kinesins were found to be nonessential for viability, but the Kinesin-13 knockout (KO) line had increased mitotic duration and reduced spindle length, whereas the Kinesin-8 KO line did not display obvious mitotic defects. Surprisingly, spindle MT poleward flux, which is mediated by Kinesin-13 in animals, was retained in the absence of Kinesin-13. MT depolymerase activity was not detectable for either kinesin in vitro, while MT catastrophe-inducing activity (Kinesin-13) or MT gliding activity (Kinesin-8) was observed. Interestingly, both KO lines showed waviness in their protonema filaments, which correlated with positional instability of the MT foci in their tip cells. Taken together, the results suggest that plant Kinesin-13 and Kinesin-8 have diverged in both mitotic function and molecular activity, acquiring roles in regulating MT foci positioning for directed tip growth.

KCBP is a microtubule (MT) minus-end-directed kinesin widely conserved in plants. It was shown in Arabidopsis that KCBP controls trichome cell shape by orchestrating MT and actin cytoskeletons using its tail and motor domains. In contrast, the KCBP knockout (KO) line in the moss Physcomitrella patens showed a defect in nuclear and organelle positioning in apical stem cells. Moss KCBP is postulated to transport the nucleus and chloroplast via direct binding to their membranes, since it binds to and transports liposomes composed of phospholipids in vitro. However, domains required for cargo transport in vivo have not been mapped. Here, we performed a structure-function analysis of moss KCBP. We found that the FERM domain in the tail region, which is known to bind to lipids as well as other proteins, is essential for both nuclear and chloroplast positioning, whereas the proximal MyTH4 domain plays a supporting role in chloroplast transport. After anaphase but prior to nuclear envelope re-formation, KCBP accumulates on the chromosomes, in particular at the centromeric region in a FERM-dependent manner. In the KCBP KO line, the rate of poleward chromosome movement in anaphase was reduced and lagging chromosomes occasionally appeared. These results suggest that KCBP binds to non-membranous naked chromosomes via an unidentified protein(s) for their transport. Finally, the liverwort orthologue of KCBP rescued the chromosome/chloroplast mis-positioning of the moss KCBP KO line, suggesting that the cargo transport function is conserved at least in bryophytes.Key words: kinesin, mitosis, chromosome segregation, kinetochore, dynein

Proper orientation of the cell division axis is critical for asymmetric cell divisions that underpin cell differentiation. In animals, centrosomes are the dominant microtubule organizing centers (MTOC) and play a pivotal role in axis determination by orienting the mitotic spindle. In land plants that lack centrosomes, a critical role of a microtubular ring structure, the preprophase band (PPB), has been observed in this process; the PPB is required for orienting (before prophase) and guiding (in telophase) the mitotic apparatus. However, plants must possess additional mechanisms to control the division axis, as certain cell types or mutants do not form PPBs. Here, using live imaging of the gametophore of the moss Physcomitrella patens, we identified acentrosomal MTOCs, which we termed “gametosomes,” appearing de novo and transiently in the prophase cytoplasm independent of PPB formation. We show that gametosomes are dispensable for spindle formation but required for metaphase spindle orientation. In some cells, gametosomes appeared reminiscent of the bipolar MT “polar cap” structure that forms transiently around the prophase nucleus in angiosperms. Specific disruption of the polar caps in tobacco cells misoriented the metaphase spindles and frequently altered the final division plane, indicating that they are functionally analogous to the gametosomes. These results suggest a broad use of transient MTOC structures as the spindle orientation machinery in plants, compensating for the evolutionary loss of centrosomes, to secure the initial orientation of the spindle in a spatial window that allows subsequent fine-tuning of the division plane axis by the guidance machinery.

Cell plate formation in plants is a complex process orchestrated by the targeted delivery of Golgi-derived and endosomal vesicles containing cell plate components to the phragmoplast midzone. It has long been hypothesized that vesicles are directionally transported along phragmoplast microtubules by motor proteins. However, the mechanisms governing the accumulation and immobilization of vesicles at the phragmoplast midzone remain elusive, and the motor protein responsible has yet to be identified. Here we show that the plant-specific class II kinesin-12 (kinesin12-II) functions as a motor protein that drives vesicle transport towards the phragmoplast midzone in the moss Physcomitrium patens . In the kinesin12-II mutant, the directional movement of cell plate materials towards the midzone and their retention were abolished, resulting in delayed cell plate formation and phragmoplast disassembly. A macroscopic phenotype arising from kinesin12-II disruption was the impediment to gametophore development. We showed that this defect was attributable to the production of aneuploid and polyploid cells in the early gametophore, where chromosome missegregation and cytokinesis failure occurred. These findings suggest that plant kinesin-12 has evolved to acquire a unique and critical function that facilitates cell plate formation in the presence of phragmoplasts. Class II kinesin-12 is responsible for transporting vesicles containing cell plate materials along phragmoplast microtubules towards the midzone, facilitating efficient cell plate formation during cytokinesis and enabling sequential cell division during multicellular organ development.

The molecular motors kinesin and dynein drive bidirectional motility along microtubules (MTs) in most eukaryotic cells. Land plants, however, are a notable exception, because they contain a large number of kinesins but lack cytoplasmic dynein, the foremost processive retrograde transporter. It remains unclear how plants achieve retrograde cargo transport without dynein. Here, we have analysed the motility of the six members of minus-end-directed kinesin-14 motors in the moss Physcomitrella patens and found that none are processive as native dimers. However, when artificially clustered into as little as dimer of dimers, the type-VI kinesin-14 (a homologue of Arabidopsis KCBP (kinesin-like calmodulin binding protein)) exhibited highly processive and fast motility (up to 0.6 μm s −1 ). Multiple kin14-VI dimers attached to liposomes also induced transport of this membrane cargo over several microns. Consistent with these results, in vivo observations of green fluorescent protein-tagged kin14-VI in moss cells revealed fluorescent punctae that moved processively towards the minus-ends of the cytoplasmic MTs. These data suggest that clustering of a kinesin-14 motor serves as a dynein-independent mechanism for retrograde transport in plants. Plants lack the retrograde motor dynein. Although kinesin-14 from Physcomitrella patens is a minus-end-directed motor, it is not individually processive. But four or more molecules acting together can transport liposomes and may substitute for dynein in plants.

Stabilisation of minus ends of microtubules (MTs) is critical for organising MT networks in land plant cells, in which all MTs are nucleated independent of centrosomes. Recently, Arabidopsis SPIRAL2 (SPR2) protein was shown to localise to plus and minus ends of cortical MTs, and increase stability of both ends. Here, we report molecular and functional characterisation of SPR2 of the basal land plant, the moss Physcomitrella patens. In protonemal cells of P. patens, where non-cortical, endoplasmic MT network is organised, we observed SPR2 at minus ends, but not plus ends, of endoplasmic MTs and likely also of phragmoplast MTs. Minus end decoration was reconstituted in vitro using purified SPR2, suggesting that moss SPR2 is a minus end-specific binding protein (-TIP). We generated a loss-of-function mutant of SPR2, in which frameshift-causing deletions/insertions were introduced into all four paralogous SPR2 genes by means of CRISPR/Cas9. Protonemal cells of the mutant showed instability of endoplasmic MT minus ends. These results indicate that moss SPR2 is a MT minus end stabilising factor.Key words: acentrosomal microtubule network, microtubule minus end, P. patens, CAMSAP/Nezha/Patronin

Cell division is essential for development and involves spindle assembly, chromosome separation, and cytokinesis. In plants, the genetic tools for controlling the events in cell division at the desired time are limited and ineffective owing to high redundancy and lethality. Therefore, we screened cell division–affecting compounds in Arabidopsis thaliana zygotes, whose cell division is traceable without time-lapse observations. We then determined the target events of the identified compounds using live-cell imaging of tobacco BY-2 cells. Subsequently, we isolated two compounds, PD-180970 and PP2, neither of which caused lethal damage. PD-180970 disrupted microtubule (MT) organization and, thus, nuclear separation, and PP2 blocked phragmoplast formation and impaired cytokinesis. Phosphoproteomic analysis showed that these compounds reduced the phosphorylation of diverse proteins, including MT-associated proteins (MAP70) and class II Kinesin-12. Moreover, these compounds were effective in multiple plant species, such as cucumber ( Cucumis sativus ) and moss ( Physcomitrium patens ). These properties make PD-180970 and PP2 useful tools for transiently controlling plant cell division at key manipulation nodes conserved across diverse plant species.

Kinesin-13 and Kinesin-8 are well-known microtubule (MT) depolymerases that regulate MT length and chromosome movement in animal mitosis. While much is unknown about plant Kinesin-8, Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) Kinesin-13 have been shown to depolymerize MTs in vitro. However, the mitotic function of both kinesins has yet to be determined in plants. Here, we generated complete null mutants of Kinesin-13 and Kinesin-8 in moss (Physcomitrella patens). Both kinesins were found to be nonessential for viability, but the Kinesin-13 knockout (KO) line had increased mitotic duration and reduced spindle length, whereas the Kinesin-8 KO line did not display obvious mitotic defects. Surprisingly, spindle MT poleward flux, which is mediated by Kinesin-13 in animals, was retained in the absence of Kinesin-13. MT depolymerase activity was not detectable for either kinesin in vitro, while MT catastrophe-inducing activity (Kinesin-13) or MT gliding activity (Kinesin-8) was observed. Interestingly, both KO lines showed waviness in their protonema filaments, which correlated with positional instability of the MT foci in their tip cells. Taken together, the results suggest that plant Kinesin-13 and Kinesin-8 have diverged in both mitotic function and molecular activity, acquiring roles in regulating MT foci positioning for directed tip growth.